Ruth Martínez-Cruz

Protein C activation peptide inhibits the expression of ICAM-1, VCAM-1, and interleukin-8 induced by TNF-a in human dermal microvascular endothelial cells

Activated protein C (APC) is generated from the cleavage of protein C by thrombin coupled to throm- bomodulin and, subsequently, is released as protein C activation peptide (papC). The aim of this study was to evaluate the effect of papC on human dermal microvascular endothelial cells (HMEC-1), activated with 5 ng/ /mL TNF-a. Flow cytometry showed that papC inhibited the expression of VCAM-1 and ICAM-1, after activa- tion with TNF-a. Similarly, RT-PCR analysis revealed that 2 and 4 pM papC inhibited the expression of VCAM-1 and IL-8 mRNA in TNF-a-treated HMEC-1. In addition, the expression of endothelial nitric oxide synthase (eNOS) increased in HMEC-1 treated with papC, compared to those without treatment. Furthermore, Jurkat cell adhesion to HMEC-1 induced by TNF-a was significantly inhibited after the addition of papC, compared to HMEC-1 without papC (p = 0.03). Finally, a control peptide analog to papC showed no effect on the expression of ICAM and VCAM on the surface of HMEC-1. In conclusion, our results suggest that papC exerts anti- inflammatory effects on endothelial cells. (Folia Histochemica et Cytobiologica 2012, Vol. 50, No. 3, 407-413)

Role of concanavalin A lectin in recognition of pterygium remnant after surgical excision: Preliminary results of a prospective study

Background: Pterygium is one of the most common conjunctival diseases among ophthalmic pathologies. The frequency of recurrences is high, either after surgical treatment or after treatment combined with mitomycin C or beta-radiation therapy. Aims: The purpose of this study was to determine whether concanavalin A (ConA) lectin bound to the pterygial surface can be used to detect recurrence or remnants of pterygium after surgical excision. Materials and Methods: This was a prospective study on 20 patients with pterygium, divided in five stages, pre-surgery, early post-surgery (24h), late post-surgery (seven days), very late post-surgery (four weeks) and two months after the procedure. A drop of fluorescein-marked Con A (35 µg/mL) was instilled in the lower conjunctival eyelid sac and the eye was exposed to the light of a Wood′s lamp for an average of five seconds. Results: Out of the 20 patients, eight patients were found to have fluorescent stretch marks over the scar corresponding to residual pterygial tissue at four weeks; two months after the procedure of re-surgery we observed no fluorescent remnants. All residual pterygia were confirmed through histochemistry studies. Conclusion: It was possible to detect remnants of pterygium in postoperative patients and recurrences in early pre-clinical stages through the visualization of fluorescent ConA bound to the pterygial surface.

Purification and Partial Characterization of β-Glucosidase in Chayote (Sechium edule).

β-Glucosidase (EC 3.2.1.21) is a prominent member of the GH1 family of glycoside hydrolases. The properties of this β-glucosidase appear to include resistance to temperature, urea, and iodoacetamide, and it is activated by 2-ME, similar to other members. β-Glucosidase from chayote (Sechium edule) was purified by ionic-interchange chromatography and molecular exclusion chromatography. Peptides detected by LC-ESI-MS/MS were compared with other β-glucosidases using the BLAST program. This enzyme is a 116 kDa protein composed of two sub-units of 58 kDa and shows homology with Cucumis sativus β-glucosidase (NCBI reference sequence XP_004154617.1), in which seven peptides were found with relative masses ranging from 874.3643 to 1587.8297. The stability of β-glucosidase depends on an initial concentration of 0.2 mg/mL of protein at pH 5.0 which decreases by 33% in a period of 30 h, and then stabilizes and is active for the next 5 days (pH 4.0 gives similar results). One hundred μg/mL β-D-glucose inhibited β-glucosidase activity by more than 50%. The enzyme had a Km of 4.88 mM with p-NPG and a Kcat of 10,000 min(-1). The optimal conditions for the enzyme require a pH of 4.0 and a temperature of 50 °C.β-Glucosidase (EC 3.2.1.21) is a prominent member of the GH1 family of glycoside hydrolases. The properties of this β-glucosidase appear to include resistance to temperature, urea, and iodoacetamide, and it is activated by 2-ME, similar to other members. β-Glucosidase from chayote (Sechium edule) was purified by ionic-interchange chromatography and molecular exclusion chromatography. Peptides detected by LC-ESI-MS/MS were compared with other β-glucosidases using the BLAST program. This enzyme is a 116 kDa protein composed of two sub-units of 58 kDa and shows homology with Cucumis sativus β-glucosidase (NCBI reference sequence XP_004154617.1), in which seven peptides were found with relative masses ranging from 874.3643 to 1587.8297. The stability of β-glucosidase depends on an initial concentration of 0.2 mg/mL of protein at pH 5.0 which decreases by 33% in a period of 30 h, and then stabilizes and is active for the next 5 days (pH 4.0 gives similar results). One hundred μg/mL β-d-glucose inhibited β-glucosidase activity by more than 50%. The enzyme had a Km of 4.88 mM with p-NPG and a Kcat of 10,000 min−1. The optimal conditions for the enzyme require a pH of 4.0 and a temperature of 50 °C.β-Glucosidase (EC 3.2.1.21) is a prominent member of the GH1 family of glycoside hydrolases. The properties of this β-glucosidase appear to include resistance to temperature, urea, and iodoacetamide, and it is activated by 2-ME, similar to other members. β-Glucosidase from chayote (Sechium edule) was purified by ionic-interchange chromatography and molecular exclusion chromatography. Peptides detected by LC-ESI-MS/MS were compared with other β-glucosidases using the BLAST program. This enzyme is a 116 kDa protein composed of two sub-units of 58 kDa and shows homology with Cucumis sativus β-glucosidase (NCBI reference sequence XP_004154617.1), in which seven peptides were found with relative masses ranging from 874.3643 to 1587.8297. The stability of β-glucosidase depends on an initial concentration of 0.2 mg/mL of protein at pH 5.0 which decreases by 33% in a period of 30 h, and then stabilizes and is active for the next 5 days (pH 4.0 gives similar results). One hundred μg/mL β-D-glucose inhibited β-glucosidase activity by more than 50%. The enzyme had a Km of 4.88 mM with p-NPG and a Kcat of 10,000 min(-1). The optimal conditions for the enzyme require a pH of 4.0 and a temperature of 50 °C.

Platelets and Platelet-Derived Microvesicles as Immune Effectors in Type 2 Diabetes

The association between type 2 diabetes mellitus (T2DM) and systemic inflammation may increase platelet reactivity and the accelerated development of vascular disease. Platelets are able to modulate the function of immune cells via the direct release of growth factors and pro-inflammatory chemokines through the production of microvesicles. The microvesicles trigger a transcellular delivery system of bioactive molecules to other cells acting as vectors in the exchange of biological information. Here, we consider the influence of platelets and platelet-derived microvesicles on cells of the immune system and the implications in the pathogenesis of T2DM.

Effects of concanavalin A on protein-C activity

Concanavalin A interacts specifically with the oligosaccharides from protein-C and modifies its anticoagulant activity. The lectin activates the protein-C activity in a dose dependent manner as demonstrated by in vitro and in vivo assays. Concanavalin A at low concentration (0.1 to 2 microg/mL) induces an increase on the catalytic activity of protein-C; at higher concentrations (5 to 20 microg/mL), the catalytic activity returns to the baseline. The effect of concanavalin A was prevented by incubating the protein-C with alpha-methyl-mannoside or by treating the purified protein-C with alpha-mannosidase; furthermore, cleavage of mannosidic residues diminishes its catalytic activity. Our results indicate that the oligomannosidic portion of protein-C participates in the regulation of the catalytic activity of this protein.